微囊藻毒素-LR對小鼠肝細胞的氧化損傷及γ-GCS基因表達的影響
發(fā)布時間：2018-08-01 08:25
【摘要】：目的：研究微囊藻毒素-LR（Microcystin-LR，MC-LR）短期重復暴露對小鼠肝細胞產生的氧化損傷效應，以及對γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthethase,γ-GCS)相關亞基的mRNA、蛋白表達水平的影響，探討MC-LR誘導的肝細胞氧化損傷與γ-GCS基因表達水平之間的關系。 方法：80只健康昆明小鼠，隨機分為2批，每批4組，每組10只，分別為對照組（含0.02%二甲基亞砜的生理鹽水0.005ml/g）、低劑量組（MC-LR5μg/kg）、中劑量組（MC-LR10μg/kg）、高劑量組（MC-LR20μg/kg），分別染毒10天和20天。每日1次經腹腔注射染毒，每隔1日稱量體重，觀察小鼠體重增長情況，計算體重增長率。分別于染毒第11天和第21天處死小鼠，取肝臟組織。用硫代巴比妥酸（TBA）法測定丙二醛（malondialdehyde，MDA）含量，用二硫代硝基苯甲酸（DTNB）法測定GSH含量；用免疫組織化學法測定DNA氧化損傷標志物8-羥基脫氧鳥苷（8-hydroxydeoxiguanosine，8-OHdG）水平；提取肝細胞RNA，逆轉錄成cDNA，用Real-time PCR法測定γ-GCS催化亞基(Glutamate cysteine ligasecatalytic subunit,GCLC)和調節(jié)亞基（Glutamate cysteine ligase modifiersubunit，GCLM）的mRNA表達水平；提取肝細胞蛋白用蛋白免疫印跡法（Western blot，WB）測定GCLC和GCLM的蛋白表達水平。 結果：1、經10天和20天染毒后，低劑量組小鼠的體重增長率與對照組比較沒有明顯差異（P0.05），中、高劑量組小鼠體重增長率均顯著低于其對照組，，差異有統(tǒng)計學意義（P0.05），且體重增長率均隨染毒劑量的增加而降低。 2、MC-LR染毒10天和20天后，低劑量染毒對小鼠肝組織MDA水平影響不明顯，差異無統(tǒng)計學意義（P0.05），而中、高劑量組MDA水平明顯高于對照組，差異有統(tǒng)計學意義（P0.05）。 3、染毒10天后，低劑量組小鼠肝組織GSH含量與對照組比較無明顯差異（P0.05），中、高劑量組GSH水平低于對照組，差異有統(tǒng)計學意義（P0.05）；染毒20天后，各劑量組GSH水平均低于對照組，有顯著性差異（P0.05）。 4、染毒10天和20天后，低、中、高劑量組小鼠肝細胞8-OHdG水平均高于對照組，差異有統(tǒng)計學意義(P0.05)，且隨著MC-LR染毒劑量的增加上升。 5、MC-LR染毒10天后，低、中、高劑量組肝細胞GCLC mRNA表達水平分別降低至對照組的72.0%、44.1%和46.5%，差異均有統(tǒng)計學意義（P0.05）；MC-LR染毒20天后，各劑量組GCLC mRNA表達水平分別低至對照組的68.5%、26.6%和26.7%，差異均有統(tǒng)計學意義（P0.05）。 6、MC-LR染毒10天后，低、中、高劑量組小鼠肝細胞GCLM mRNA表達水平明顯低于對照組，分別降至對照組的59.4%、42.5%和34.3%，差異有統(tǒng)計學意義（P0.05）；染毒20天結果顯示，低、中、高劑量組GCLMmRNA表達水平分別降低至對照組的62.9%、35.8%和39.0%（P0.05）。 7、MC-LR染毒10天及20天，低劑量組小鼠肝細胞GCLC蛋白表達量均無顯著性變化（P0.05），但中、高劑量組GCLC蛋白表達量均明顯低于對照組，差異有統(tǒng)計學意義（P0.05）；染毒20天后，低、中、高劑量組GCLC蛋白表達量均低于染毒10天相應的劑量組，差異有統(tǒng)計學意義（P0.05）。 8、染毒10天后，低劑量組小鼠肝細胞GCLM蛋白表達量與對照組比較無顯著性差異，中、高劑量組GCLM蛋白表達水平均明顯低于對照組，差異有統(tǒng)計學意義（P0.05）；染毒20天后，低、中、高劑量組GCLM蛋白表達水平均低于對照組，差異有統(tǒng)計學意義（P0.05）；中劑量組染毒20天后，GCLM蛋白表達水平明顯低于染毒10天的水平，差異有統(tǒng)計學意義（P0.05）。 9、肝細胞GCLC、GCLM mRNA表達水平與MDA水平呈正相關關系（rGCLC=0.425，P0.05；rGCLM=0.304，P0.05）；肝細胞GCLC、GCLM mRNA表達水平與8-OHdG水平呈正相關關系（rGCLC=0.420，P0.05；rGCLM=0.476，P0.05）。結論： MC-LR可抑制小鼠肝細胞γ-GCS的催化亞基GCLC、調節(jié)亞基GLCM的mRNA表達和蛋白表達，降低GSH水平，升高MDA和8-OHdG水平，引起脂質和DNA氧化損傷，但MC-LR對γ-GCS表達的影響機制仍有待于進一步研究。
[Abstract]:Objective: To study the oxidative damage effect of microcystin -LR (Microcystin-LR, MC-LR) on mice liver cells, and the effect on the mRNA and protein expression level of gamma glutamyl cysteine synthetase (gamma -glutamylcysteine synthethase, gamma -GCS) related subunits, and to explore the oxidative damage of hepatocytes and gamma -GCS induced by MC-LR. The relationship between the level of gene expression.
Methods: 80 healthy Kunming mice were randomly divided into 2 batches, each group of 4 groups, 10 in each group, the control group (including 0.02% two methyl sulfoxide 0.005ml/g), the low dose group (MC-LR5 mu g/kg), the medium dose group (MC-LR10 mu g/kg), the high dose group (MC-LR20 mu g/kg), respectively for 10 days and 20 days, 1 times a day by intraperitoneal injection, and every 1 days. The weight growth of mice was observed and the growth rate of body weight was calculated. The mice were killed for eleventh days and twenty-first days and the liver tissues were taken respectively. The content of malondialdehyde (malondialdehyde, MDA) was determined by thiobarbituric acid (TBA) method and the content of GSH was measured with two thiobenzoic acid (DTNB) method. The oxidative damage standard of DNA was measured by immunohistochemistry. The level of 8- hydroxy deoxy guanosine (8-hydroxydeoxiguanosine, 8-OHdG) was extracted, RNA of liver cells was extracted and cDNA was reverse transcriptase, and the expression level of gamma -GCS catalytic subunit (Glutamate cysteine ligasecatalytic subunit) and regulating subunit was extracted by Real-time PCR method; the liver cell eggs were extracted. The protein expression levels of GCLC and GCLM were measured by Western blot (WB).
Results: 1, after 10 days and 20 days of poisoning, the weight growth rate of mice in low dose group was not significantly different from that of the control group (P0.05). In the high dose group, the weight growth rate of the mice was significantly lower than that of the control group, the difference was statistically significant (P0.05), and the weight growth rate decreased with the increase of the dose.
2, after 10 and 20 days of MC-LR poisoning, the effect of low dose on the MDA level of liver tissue in mice was not significant (P0.05), but in the high dose group, the level of MDA was significantly higher than that in the control group, the difference was statistically significant (P0.05).
3, after 10 days of poisoning, the GSH content in the liver tissue of the low dose group was not significantly different from that of the control group (P0.05). In the high dose group, the GSH level was lower than the control group, the difference was statistically significant (P0.05), and the GSH level in each dose group was lower than that of the control group for 20 days, and there was a significant difference (P0.05).
4, after 10 and 20 days, the levels of 8-OHdG in the liver cells of the low, middle and high dose groups were all higher than those of the control group, the difference was statistically significant (P0.05), and increased with the increase of the dose of MC-LR.
5, after 10 days of MC-LR poisoning, the expression level of GCLC mRNA in liver cells in low, middle and high dose groups decreased to 72%, 44.1% and 46.5% in the control group, and the difference was statistically significant (P0.05). After 20 days of MC-LR, the expression level of GCLC mRNA in each dose group was 68.5%, 26.6% and 26.7% in the control group respectively. The difference was statistically significant (P0.05).
6, after 10 days of MC-LR poisoning, the expression level of GCLM mRNA in the hepatocytes of low, middle and high dose mice was significantly lower than that of the control group, which decreased to 59.4%, 42.5% and 34.3% of the control group, respectively (P0.05). The GCLMmRNA expression level in the low, middle and high dose groups decreased to 62.9%, 35.8% and 39% (P0.05) in the lower, middle and high dose groups respectively.
7, MC-LR was poisoned for 10 days and 20 days, and there was no significant change in the expression of GCLC protein in the liver cells of the low dose group (P0.05), but the expression of GCLC protein in the high dose group was significantly lower than that of the control group. The difference was statistically significant (P0.05). The expression of GCLC protein in the low, middle and high dose group was lower than that of the corresponding dose group of the 10 day poisoned group, and the difference was lower than that of the corresponding dose group for 10 days. There was a statistical significance (P0.05).
8, after 10 days of poisoning, there was no significant difference in the expression of GCLM protein in the liver cells of the low dose group with the control group, and the expression level of GCLM protein in the high dose group was significantly lower than that of the control group (P0.05), and the expression level of GCLM protein in the low, middle and high dose group was lower than that of the control group for 20 days, and the difference was statistically significant (P The expression of GCLM protein in middle dose group was significantly lower than that in 10 days after exposure (P 0.05).
9, the expression level of GCLC and GCLM mRNA was positively correlated with the level of MDA (rGCLC=0.425, P0.05; rGCLM=0.304, P0.05), and the expression level of the hepatocyte GCLC and GCLM mRNA was positively correlated with the 8-OHdG level. The expression of mRNA and protein, decrease the level of GSH and increase the level of MDA and 8-OHdG, cause the oxidative damage of lipid and DNA, but the mechanism of MC-LR on the expression of gamma -GCS remains to be further studied.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575
本文編號：2156968
[Abstract]:Objective: To study the oxidative damage effect of microcystin -LR (Microcystin-LR, MC-LR) on mice liver cells, and the effect on the mRNA and protein expression level of gamma glutamyl cysteine synthetase (gamma -glutamylcysteine synthethase, gamma -GCS) related subunits, and to explore the oxidative damage of hepatocytes and gamma -GCS induced by MC-LR. The relationship between the level of gene expression.
Methods: 80 healthy Kunming mice were randomly divided into 2 batches, each group of 4 groups, 10 in each group, the control group (including 0.02% two methyl sulfoxide 0.005ml/g), the low dose group (MC-LR5 mu g/kg), the medium dose group (MC-LR10 mu g/kg), the high dose group (MC-LR20 mu g/kg), respectively for 10 days and 20 days, 1 times a day by intraperitoneal injection, and every 1 days. The weight growth of mice was observed and the growth rate of body weight was calculated. The mice were killed for eleventh days and twenty-first days and the liver tissues were taken respectively. The content of malondialdehyde (malondialdehyde, MDA) was determined by thiobarbituric acid (TBA) method and the content of GSH was measured with two thiobenzoic acid (DTNB) method. The oxidative damage standard of DNA was measured by immunohistochemistry. The level of 8- hydroxy deoxy guanosine (8-hydroxydeoxiguanosine, 8-OHdG) was extracted, RNA of liver cells was extracted and cDNA was reverse transcriptase, and the expression level of gamma -GCS catalytic subunit (Glutamate cysteine ligasecatalytic subunit) and regulating subunit was extracted by Real-time PCR method; the liver cell eggs were extracted. The protein expression levels of GCLC and GCLM were measured by Western blot (WB).
Results: 1, after 10 days and 20 days of poisoning, the weight growth rate of mice in low dose group was not significantly different from that of the control group (P0.05). In the high dose group, the weight growth rate of the mice was significantly lower than that of the control group, the difference was statistically significant (P0.05), and the weight growth rate decreased with the increase of the dose.
2, after 10 and 20 days of MC-LR poisoning, the effect of low dose on the MDA level of liver tissue in mice was not significant (P0.05), but in the high dose group, the level of MDA was significantly higher than that in the control group, the difference was statistically significant (P0.05).
3, after 10 days of poisoning, the GSH content in the liver tissue of the low dose group was not significantly different from that of the control group (P0.05). In the high dose group, the GSH level was lower than the control group, the difference was statistically significant (P0.05), and the GSH level in each dose group was lower than that of the control group for 20 days, and there was a significant difference (P0.05).
4, after 10 and 20 days, the levels of 8-OHdG in the liver cells of the low, middle and high dose groups were all higher than those of the control group, the difference was statistically significant (P0.05), and increased with the increase of the dose of MC-LR.
5, after 10 days of MC-LR poisoning, the expression level of GCLC mRNA in liver cells in low, middle and high dose groups decreased to 72%, 44.1% and 46.5% in the control group, and the difference was statistically significant (P0.05). After 20 days of MC-LR, the expression level of GCLC mRNA in each dose group was 68.5%, 26.6% and 26.7% in the control group respectively. The difference was statistically significant (P0.05).
6, after 10 days of MC-LR poisoning, the expression level of GCLM mRNA in the hepatocytes of low, middle and high dose mice was significantly lower than that of the control group, which decreased to 59.4%, 42.5% and 34.3% of the control group, respectively (P0.05). The GCLMmRNA expression level in the low, middle and high dose groups decreased to 62.9%, 35.8% and 39% (P0.05) in the lower, middle and high dose groups respectively.
7, MC-LR was poisoned for 10 days and 20 days, and there was no significant change in the expression of GCLC protein in the liver cells of the low dose group (P0.05), but the expression of GCLC protein in the high dose group was significantly lower than that of the control group. The difference was statistically significant (P0.05). The expression of GCLC protein in the low, middle and high dose group was lower than that of the corresponding dose group of the 10 day poisoned group, and the difference was lower than that of the corresponding dose group for 10 days. There was a statistical significance (P0.05).
8, after 10 days of poisoning, there was no significant difference in the expression of GCLM protein in the liver cells of the low dose group with the control group, and the expression level of GCLM protein in the high dose group was significantly lower than that of the control group (P0.05), and the expression level of GCLM protein in the low, middle and high dose group was lower than that of the control group for 20 days, and the difference was statistically significant (P The expression of GCLM protein in middle dose group was significantly lower than that in 10 days after exposure (P 0.05).
9, the expression level of GCLC and GCLM mRNA was positively correlated with the level of MDA (rGCLC=0.425, P0.05; rGCLM=0.304, P0.05), and the expression level of the hepatocyte GCLC and GCLM mRNA was positively correlated with the 8-OHdG level. The expression of mRNA and protein, decrease the level of GSH and increase the level of MDA and 8-OHdG, cause the oxidative damage of lipid and DNA, but the mechanism of MC-LR on the expression of gamma -GCS remains to be further studied.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575
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